Foxp3+ regulatory T cells, or ‘Tregs’ are fundamental in controlling various immune responses in that Tregs can rapidly suppress the activity of other immune cells. In particular, Tregs are crucial for maintaining tolerance by downregulating undesired immune responses to self and non-self antigens (see, e.g. Fontenot, J. D. & Rudensky, A. Y. Nat Immunol 6, 331-7 (2005); Sakaguchi, S., Annu Rev Immunol 22, 531-62 (2004)). For instance, Treg defects have been discovered in patients with multiple sclerosis (MS), type I diabetes (T1D), psoriasis, myasthenia gravis (MG) and other autoimmune diseases (Baecher-Allan, C. & Hafler, D. A., Immunol Rev 212, 203-16 (2006)). Similar links may also exist for atopy and allergic diseases (Robinson, D. S., Larche, M. & Durharn, S. R., J Clin Invest 114, 1389-97 (2004)). For all these diseases reports exist pointing to a reduced in vitro immune suppression of the patient's Treg cells. This has led to an increasing interest in the possibility of using Tregs in immunotherapy to treat or prevent chronic infections, autoimmune diseases, allergies and transplantation-related complications, such as graft rejection or graft-versus-host disease (GvHD) (For a review, see Roncarolo, M. G. & Battaglia, M., Nat Rev Immunol 7, 585-98 (2007)).
Treg cells constitute of about 2-10% of CD4+ T cells in humans and rodents and constitutively express CD25, CTLA-4 and GITR, as well as the transcription factor Foxp3, which is involved in their development and function. The characteristic marker of Treg cells is Foxp3. Methods for the isolation of human Foxp3+ Treg cells are known. For instance, Hoffmann, P. et al. Biol Blood Marrow Transplant 12, 267-74 (2006) describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol under good manufacturing practice (GMP) conditions). The generated cell products contained on average 49.5% Foxp3+ Treg cells. Also, commercial kits, e.g. CD4+CD25+ Regulatory T Cell Isolation Kit from Miltenyi Biotec or DYNAL® CD4+CD25+ Treg Kit from Invitrogen are available.
All of the hitherto described methods for isolation of human Foxp3+ Treg cells employ positive selection of Foxp3+ Treg cells based on cell surface markers of Tregs (see, e.g. Seddiki, N. et al., J Exp Med 203, 1693-700 (2006)). That is, the Foxp3+ Treg cells are isolated by using antibodies for Treg associated cell surface markers, mostly CD25. Yet most cell surface markers of Tregs, such as CD4 and CD25, are not restricted to Tregs. For instance, the commonly employed CD25 is not present on all Foxp3+ Treg cells and is also expressed by effector and memory CD4+ T cells (see, e.g. Baecher-Allan, C., Brown, J. A., Freeman, G. J. & Hafler, D. A., J Immunol 167; 1245-53 (2001)). Consequently, these positive selection methods do not permit the isolation of a uniform population that accounts for most of the Foxp3+ Treg cells as outlined above; Hoffmann, P. et al. obtained on average 49.5% Foxp3+ Treg cells.
Another disadvantage of current methods is the contamination of the isolated Treg subsets with effector T cells. The latter represent an inherent risk of adverse reactions as they drive pro-inflammatory immune reactions by secreting cytokines such as IFN-γ or IL-17. When employing markers such as CD25 these contaminations can be significant as up to half of the isolated CD4+ cell population can be comprised of effector T cells (Hoffmann, P. et al. Biol Blood Marrow Transplant 12, 267-74 (2006)).
Also, application of Foxp3+ Treg cells that have been isolated by positive selection based on cell surface markers of Tregs, in cellular therapy poses severe problems. First, isolation of Foxp3+ Treg cells based on cell surface markers, e.g. CD25, leads to more or less severe contaminations of the Foxp3+ Treg cell population with other cells, e.g. CD4+ effector cells which represent the main target of Treg suppression. Accordingly, there is a high risk if such positively selected Foxp3+ Treg cells were to be applied in cellular therapy as it might lead to a potentially fatal activation of the immune system of the patient treated. Such a fatal immune response was recently documented in the failure of the ‘Tegenero’ trials, where an antibody expected to expand Treg cells led to an activation of effector cells (Suntharalingam, G. et al., N Engl J Med 355, 1018-28 (2006)). Second, positively selected Foxp3+ Treg cells which have been tagged by an antibody may exhibit impaired function. For instance, cells tagged by an antibody are potentially preactivated, may suffer complementor cell-mediated depletion or exhibit altered homing and migration patterns. Accordingly, Foxp3+ Treg cells targeted by antibodies during their isolation are undesirable for a therapeutic use not only for safety reasons.
As discussed, the methods and kits described above show major disadvantages with respect to isolating Foxp3+ Treg cells. Firstly, the current methods for the isolation of immune-suppressive Foxp3+ Treg cells do not allow an effective removal of contaminating CD4+ effector and memory T cells. This is because the currently employed techniques and markers, e.g. CD25-based Foxp3+ Treg isolations, fail to discriminate these contaminating CD4+ effector and memory T cells from immune-suppressive Foxp3+ Treg cells. For instance, CD25 is a marker that is also present on these contaminating CD4+ cells. Moreover, at least some of these contaminating CD4+ cells cannot even be discriminated by intracellular Foxp3 staining, since activated human CD4+ effector cells are known to express Foxp3 transiently (Allan et al., Int Immunol. 19:345-54(2007)). As a result, even highly pure populations of CD25highCD4+ T cells isolated by current methods contain a substantial fraction of cytokine producing pro-inflammatory effector cells (Dieckmann et al., J. Exp Med. 193, 1303-1310 (2001)), i.e. the isolated Foxp3+ Treg cells are significantly contaminated with CD4+ effector and memory T cells.
Additionally, so far no method exists that allows access to Foxp3+ Treg cells by negative selection, i.e. leaving the Foxp3+ Treg cells label/antibody-free.
From the foregoing it follows that there is a particular need for methods and kits/compositions useful for isolating Foxp3+ Treg cells which are virtually free from CD4+ effector and memory T cells. There is also a particular need for methods that do not require positive selection of Foxp3+ Treg cells.